Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-05-28 till 2009-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study in accordance with GLP standard procedure.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Sodium Hypophosphite; tested in the form of monohydrate as the anhydrous form (CAS 7681-53-0) is highly hygroscopic and difficult to handle without specific precautions.
- Physical state: white crystalline solid
- Supplier: Rhodia UK-Oldbury
- Batch number: 90113D
- Expiry date: June 2010
- Storage condition of test material: room temperature, stored in a tightly closed container, opening of the container restricted to the minimum needs
- Purity: 99.5%
- Impurities (identity and concentrations):
Phosphite (Na2HPO3): 0.14%
Calcium (Ca): 21 ppm
Iron (Fe): 0.7 ppm
Sulphate (SO4): 8 ppm
Chloride (Cl): 64 ppm
- Certificate of analysis:
Analysis number: ITC/09/01/04
Analysis date: 2009-01-27

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks
- Weight at study initiation (± SD): 21.4 ± 1.0 g
- Housing: individually in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before beginning of the study
- Other:
nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): ± 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: Death on 2009-06-08.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0 (control), 2.5, 5, 10, 25 or 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TEST TO ASSESS IRRITANT POTENTIAL THROUGH EAR THICKNESS MEASUREMENT:
- Compound solubility: Due to the unsatisfactory solubility of the test item in the first recommended vehicles (acetone/olive oil (4/1,v/v), dimethylformamide and methyl ethyl ketone), propylene glycol was chosen from the other proposed vehicles. A homogeneous suspension was obtained at the maximum concentration of 50%. Hence concentrations for the preliminary test were 5, 10, 25 and 50%
- Irritation: The irritant potential was assessed through ear thickness measurement. The test item was non-irritant in the preliminary test, whatever the concentration. The highest concentration retained for the main test was therefore the maximal practicable concentration, according to the criteria specified in the International Guidelines.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of the test method: murine Local Lymph Node assay (LLNA)
- Criteria used to consider a positive response:
The test item was considered as a skin sensitizer when the StimuIation indices (SI) for a dose group is >= 3. Other relevant criteria such as cellularity and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION
Each dosage form preparation was prepared each day separately from each other and homogenized by magnetic stirring.

ADMINISTRATION:
On days 1, 2 and 3, 25 µL of the control or dosage form preparations (i.e. test substance or reference substance) was applied to the dorsal surface of both ears. To avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the pipette's tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
In the positive control group given hexyl cinnamic aldehyde (concentration 25%) a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 15.30) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See table 1 in section remarks on results including tables and figures
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table 1 in section remarks on results including tables and figures

Any other information on results incl. tables

- Clinical examinations

- Systemic clinical signs and mortality: Neither mortality nor clinical signs were observed.

- Body weight: no change in body weight compared to the control group.

- Local irritation: Neither cutaneous reactions nor notable increase in ear thickness were observed in the animals of the treated groups.

 

- Proliferation assay

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of3H-TdR.

The cell viability was higher than 70% in each group. 

No notable lymphoproliferation was noted at any of the tested concentrations of the test substance.

- Table 1: Detailed overview of the study results

Treatment

Conc

(%)

Cell viability

Cellularity index

# of nodes per group

dpm per group

dpm per node

Stimulation Index

Increase in ear thickness

(% between day 1 and 6)

Irritation level
(based on increase in ear thickness)

Vehicle

 

90.91

 

8

891.00

111.38

 

0

 

Test substance

2.5

90.14

1.60

8

1594.54

199.32

1.79

5.15

non-irritant

Test substance

5

82.14

1.15

8

898.61

112.33

1.01

3.03

non-irritant

Test substance

10

96.97

0.80

8

1099.35

137.42

1.23

2.04

non-irritant

Test substance

25

95.65

1.10

8

1185.10

148.14

1.33

4.04

non-irritant

Test substance

50

76.19

1.20

8

1268.55

158.57

1.42

2.94

non-irritant

Positive control

25

94.92

8.40

8

13631.16

1703.90

15.30

 

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item sodium hypophosphite did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The potential of sodium hypophosphite to induce delayed contact hypersensitivity was assessed in mice according to the OECD guideline 429 and to the EU Method B.42. The study was conducted in compliance with the principles of Good Laboratory Practice regulations on the monohydrated form of the test item as the anhydrous form is highly hygroscopic and difficult to handle without specific precautions.

Five groups of 4 animals were exposed to the test substance on 3 consecutive days (concentrations 2.5%, 5%, 10%, 25% and 50%) in parallel with a negative control group receiving the vehicle propylene glycol and a positive control group receiving the moderate sensitiser alpha-hexylcinnamaldehyde. After 2 days of resting, the animals were sacrificed in order to perform the proliferation assay. Changes in body weight, ear thickness were noted and the animals were checked on a regular base for clinical signs, mortality and local irritation.

A significant lymphoproliferation was noted in the positive control given alpha-hexylcinnamaldehyde. The study was therefore considered valid. No noteworthy lymphoproliferation was noted with the test substance at any tested concentration. The Stimulation Index was below 3 (range: 1.01 to 1.79) and no dose response reaction was observed.

No systemic clinical signs and mortality, neither changes in body weight, nor local irritation was observed for all tested concentrations. As a result, sodium hypophosphite was considered as not sensitising.